Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination

Abstract

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

Graphical Abstract

Rad51 inhibits DNA synthesis by Pold (use Greek delta for last letter) during Rad52-mediated repeat recombination. Poll (use Greek lambda for last letter) can displace Rad51 to allow PCNA loading and Pold (use Greel for last letter) DNA synthesis.

Figure 1.

Loss of Pol4 activity confers ionizing radiation (IR) sensitivity. (A–D) Cultures of haploid WT and mutant cells were grown to mid-log, counted by hemocytometer, and spread onto YPD plates before exposure to 0, 10, 50, 100 or 150 Grays of ionizing radiation. Percent survival was determined for each genotype by counting the number of colonies, dividing by the number of cells plated, and multiplying by 100. The mean percent survival ± SEM was determined for each genotype from a minimum of 9 independent cultures, normalized to cells not exposed to IR, and plotted against radiation exposure in Grays. (A) Percent survival of pol4Δ (haploid segregant of WDHY2816), pol4-CD (haploid segregant of WDHY2895), dnl4Δ (haploid segregant of WDHY2829), and pol4Δ dnl4Δ (haploid segregant of WDHY3150) mutants compared to WT (haploid segregant of WDHY2990) and rad52 (WDHY2556) following varying levels of IR exposure. (B–D) IR survival curves for WT (haploid segregant of WDHY2990) and pol4Δ (haploid segregant of WDHY2816) mutant cells possessing an extrachromosomal vector with a functional yeast POL4 (pWDH1145) or human hPOLL (pWDH1146) DNA polymerase λ gene, a DNA polymerase defective allele from yeast, pol4-CD (pWDH1147) or human hPOLL-CD (pWDH1148), or without an insert, empty vector (pWDH403). The absence of error bars indicates that they are smaller than the plotting symbol.

Figure 2.

Direct-repeat recombination rate is influenced by Pol4 and Rad51. (A) Spontaneous direct-repeat recombination is measured as the rate of His⁺ recombinant formation in haploid cells possessing two truncated his3 alleles sharing 103 bp or 415 bp of homology. (B) Haploid wildtype (WT) cells (WDHY5901), rad52Δ (WDHY5905), rad52Δ pol4Δ (WDHY6173), pol4Δ (WDHY5909), pol4-CD (WDHY5843), rad51Δ (segregant of WDHY5227), rad51Δ pol4Δ (segregant of WDHY5226), and rad51Δ pol4-CD (segregant of WDHY5226) mutants show varying direct-repeat recombination rates using 103 bp of homology. (C) Haploid wildtype (WT) cells (WDHY5903), rad52Δ (WDHY5907), rad52Δ pol4Δ (WDHY6174), pol4Δ (WDHY5911), pol4-CD (WDHY5845), rad51Δ (segregant of WDHY5224), pol4Δ rad51Δ (segregant of WDHY5225), and pol4-CD rad51Δ (segregant of WDHY5225) mutants show varying direct-repeat recombination rates using 415 bp of homology. The median rate and ± 95% confidence interval of direct-repeat recombination was calculated from a minimum of nine independent cultures. * indicates significant differences as determined using pair-wise unpaired t-tests with P ≤ 0.05. The fold increases/decreases are given for the comparison indicated by the P-value with the exception of the comparison for pol4Δ and pol4-CD in C, for which no fold change is given.

Figure 3.

Pol4 and Polδ cooperate to overcome Rad51 inhibition of DNA synthesis. (A) DNA synthesis assay using substrates olWDH2123 (80mer) and olWDH2124 (35mer). Black dots indicate biotin labels at the 5’ and 3’ ends of the 80mer. (B) Denaturing 10% acrylamide gel of DNA synthesis reactions. Reactions were carried out with 25 nM molecules substrate incubated without or with Rad51 (1:12 nt, 1:6 nt, 1:3 nt, 1:1 nt of the 80mer) and 12.5 nM of Polδ. (C and D) Quantitation of results in (B) and additional experiments. Data shows mean ± SEM of three independent experiments. (E) Denaturing 10% acrylamide gel of DNA synthesis reactions. Reactions were carried with 25 nM molecules substrate incubated without or with Rad51 (1:1 nt) or Pol4 (12.5, 25, 50,100 nM) or Pol4-CD (100 nM) or Polδ (12.5 nM). The dotted line and black triangle indicate that lanes 8 and 9 were taken from a different gel. (F and G) Quantitation of results in (E) and additional experiments. Data show mean ± SEM of n = 3. * denotes bands below the primer that derive from shorter oligonucleotide synthesis products and products of Polδ exonuclease activity.

Figure 4.

Rad51 inhibition of annealing-dependent DNA synthesis. (A) Reaction schemes for DNA synthesis with pre-annealed (see Figure 3) and not pre-annealed primer. Black dots indicate biotin labels at the 5’ and 3’ ends of the 80mer. (B) Reactions were carried out in the presence of 25 nM of the 35mer substrate and 25 nM of the 80mer. Black dots indicate biotin labels at the 5’ and 3’ ends of the 80mer. Proteins were added as follows: ± Rad51 (1:3 nt), ± Pol4 (12.5, 25, 50 nM) or Pol4-CD (50 nM) and ± 12.5 nM of Polδ. (C) Quantitation of DNA synthesis inhibition by Rad51 in reactions with pre-annealed primers (Figure 3B, lanes 2, 5), without pre-annealed primers (B, lanes 2, 3) and additional replica experiments. Reactions contained Rad51 at 1:3 nt. Data shows mean ± SEM of n = 3.

Figure 5.

Pol4 suppresses Rad51 inhibition of annealing-dependent DNA synthesis in the presence of Rad51 and PCNA/RFC. (A) Annealing-dependent DNA synthesis assay. (B) Denaturing 10% acrylamide gel of DNA synthesis reactions. The broken line indicates that the control lanes (1,2) were from the same gel with intervening lanes omitted. Lanes 6–8 were from a different gel. Reactions were carried in the presence of 50 nM of the 35mer substrate and 50nM of the 80mer. Proteins were added as follows: ± Rad51 (1:9 nt), ± RPA (1:60 nt), Rad52 (1:40 nt), ± Pol4 (25 nM), ± PCNA/RFC (50 nM each) and ± 25 nM of Pol δ and (C) Quantitation comparing the Pol4 effect on DNA synthesis in the presence of RPA and Rad52 in reactions without Rad51 (B, lanes 4 and 5) and with Rad51 (B, lanes 7 and 8) and additional replica experiments. The data reflect the increase (fold-change) in Polδ synthesis in the presence of Pol4 normalized by the respective reaction without Pol4. Data show mean ± SEM of n = 3. * marks bands that arise from secondary structures of the primer; see lane 1 in (B).

Figure 6.

Pol4 displaces Rad51 from DNA. (A) Rad51 displacement assay. Black dots indicate biotin labels at the 3’ end of the 80mer. (B) Immunoblots of bound and unbound Rad51 and Pol4 or Pol4-CD. Reactions analyzed were carried out with 50 nM of the annealed tailed DNA substate, 375 nM of Rad51 (1:6 nt of the ssDNA part of the tailed DNA), and increasing amount of Pol4 or Pol4-CD (0, 25, 50, 100 and 200 nM). (C) Quantitation of results in (B) and additional experiments. Data show mean ± SEM of n = 3.

Figure 7.

Pol4 catalytic activity is required in the presence of a + 1 nt flap at the 3’-OH end. (A) Annealing-dependent DNA synthesis assay. Black dots indicate biotin labels at the 5’ and 3’ ends of the 80mer. (B) Denaturing 10% acrylamide gel of DNA synthesis reactions. Reactions were carried out in the presence of 25 nM of the 34 + 1mer substrate and 25 nM of the 80mer. Proteins were added as follows: ± Rad51 (1:3 nt), ± PCNA/RFC (25 nM each), ± Pol4 (12.5, 25, 50 nM) or Pol4-CD (50 nM) and ± 12.5 nM of Polδ. Broken lines indicate that some lanes from the gels were spliced out. (C) Quantitation of DNA total synthesis (see Material and Methods) from (B) and additional experiments. Data show mean ± SEM of 3 independent experiments.